Purpose:
To determine the biological significance of HTT upregulation on retinal cell survival in diabetes.

Methods:
Human donor globes of 3 patients with non-proliferative diabetic retinopathy were obtained within 6 hours of death. Sensory retina was dissected and fixed. Tissue blocks were embedded in paraffin and immunostained for the expression of HTT (rabbit anti-human, 1:1000, Sigma) and polyQ repeats (mouse anti-human, 1:100, Millipore). TUNEL staining was used to identify apoptotic cells. Samples were examined using a confocal microscope (Olympus FluoViewTM FV1000). Co-localization of HTT with polyQ repeats and TUNEL signal was quantified as a percentage of the whole photoreceptor cell population. Retinal sections obtained from age-matched fresh non-diabetic (n=3) individuals were used as controls. Similar associations were sought in female and male heterozygous C57BL/6-Ins2Akita/J mice that develop mild and severe hyperglycemia, respectively. Normoglycemic C57BL/6J animals were used as controls.

Results:
In non-diabetic human eyes HTT is expressed mainly in the inner retina. Occasional punctuate cytoplasmic polyQ aggregates were seen within ganglion cells. In diabetic eyes, HTT expression increases mainly in the outer retina with a marked shift of HTT towards the outer segments, where it is normally present only in minute amounts. Diabetes is characterized by perinuclear polyQ aggregates of HTT in the outer nuclear layer as well as in ganglion cells. In diabetic human eyes 4.40 ± 2.69 % of the photoreceptors were stained positive for TUNEL whereas no TUNEL (+) cells were noted in the retina of non-diabetic donors. Apoptotic signal was almost always coinciding with the presence of perinuclear HTT aggregates in photoreceptor cells. Similarly, hyperglycemia induced HTT upregulation in the photoreceptors and its migration towards the outer segments in C57BL/6-Ins2Akita/J mice. HTT expression coincided with increased photoreceptor cell apoptosis (1.90 ± 0.99 %) which was not observed in normoglycemic animals.

Conclusions:
In diabetic retina, HTT is upregulated in the outer retina prior to the development of any structural lesions. HTT perinuclear aggregation is associated with photoreceptor cell apoptosis. Identification of the early photoreceptor death mechanisms can create new opportunities for drug development to prevent vision loss in diabetes.